Diabetes is a growing epidemic, and the increasing number of type 2 diabetics highlights the need for novel, mechanistically distinct, therapeutics. In this application, the potential of insulin-degrading enzyme (IDE) inhibitors as diabetes drugs will be explored. Preceding work led to the discovery of 6bK, a small-molecule macrocycle IDE inhibitor. 6bK is a remarkably selective IDE inhibitor with greater than 1000-fold selectivity over related metalloproteases. Structural studies determined that this selectivity comes from 6bK binding to a unique site on the IDE surface (referred to as the IDE-selectivity pocket). Though other IDE inhibitors have been reported, 6bK is the only IDE inhibitor that is active in vivo. Usin 6bK, the physiological impact of IDE inhibition in mouse models of diabetes (HFD-fed mice) was tested and 6bK-treated mice had higher insulin levels and improved oral glucose tolerance compared to vehicle-treated animals. Furthermore, these experiments revealed that the pancreatic hormones amylin and glucagon are also endogenous IDE substrates. Pramlintide, an amylin analog and amylin receptor agonist, is an FDA-approved drug that slows gastric emptying to control postprandial blood glucose levels. This information was used to demonstrate that 6bK slows gastric emptying by raising endogenous amylin levels, providing a second signaling pathway that 6bK uses to lower blood glucose levels. IDE inhibitors are worth further investigation because they represent a mechanistically distinct class of potential diabetes drugs; the only compounds that raise insulin and amylin levels by impairing the degradation of these hormones. Medicinal chemistry, structural biology, and physiology will be used to determine the clinical potential of IDE inhibitors. First, 6bK will be used to determine whether chronic IDE inhibition is a safe and effective treatment of diabetes in animal models (Aim 1). Next, novel IDE inhibitors that were designed using structure-activity-relationships, structural biology, and computation will be generated and tested (Aim 2). Finally, IDE inhibitors will be used in combination with approved diabetic drugs to identify synergistic effects on lowering blood glucose levels (Aim 3). These experiments might reveal combinations of compounds, including IDE drugs, that will be most useful clinically. Together these studies should provide the clearest picture of the clinical potential of IDE inhibitors, and reveal compounds that can serve as useful lead compounds for drug development.